Re: [ccp4bb] Adding Zinc to Protein

[Artem Evdokimov]

Dear Nicola,

Happy New Year

It may or may not be possible to re-constitute Zn into purified protein.
Some binding sites are very hard to fill back up once Zn is gone (for
example sites with more than one -SH ligand can and often do undergo
oxidative crosslinking in the absence of a stabilizing ion - we ran into
this plenty of times when working with Zn-finger proteins). Other sites
(e.g. Zn proteases) can easily re-gain Zn that has been stripped away (a
good method for avoiding autoproteolysis when purifying certain
metalloenzymes, but it can also backfire horribly). Note that common
reducing agent DTT is know to strip Zn and other transition elements out of
protein (BME is much less likely to do this and TCEP is virtually safe from
this perspective). Ditto common buffers - citrate, high phosphate
(especially with Iron ions), malonate, imidazole and so on.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133908/

Please note that Zn complex chemistry in water is "...fundamental, rather
complex, and has important consequences for the role of zinc in biology..."
to quote Krezel & Maret (reference below) - so if reconstitution does not
work in some conditions it very well might work in others.

https://www.sciencedirect.com/science/article/pii/S0003986116301308

So if your protein lost its Zn, there is no really good way to predict the
outcome of an attempted re-fill -- but since you have frozen protein in
spades your best bet is to try a few things, such as dialyzing a small
sample of protein (ideally diluted to ~1mg/ml or so) against low
concentration of Zn in a suitable buffer. A good place to start is ~10uM
(that's micromolar) Zn - which is plenty to saturate the protein with
reasonable binding constant but is (hopefully) not enough to precipitate
the protein out. After dialysis, remove Zn from solution - by briefly
re-dialyzing against Zn-free bufffer - then concentrate the protein and try
it out. You can also attempt to stabilize Zn in solution with an equimolar
(or slightly less than equimolar) amount of a mild chelator like malonate
(not EDTA, its complex with Zn is far too stable) and then add it directly
to your protein - note that adding Zn to protein directly tends to cause
precipitation (but chelating the Zn with a moderate-strength agent tends to
prevent this).

Good luck,

Artem
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On Sun, Dec 9, 2018 at 4:32 PM Nicola Evans <
251ca3b4615e-dmarc-requ...@jiscmail.ac.uk> wrote:

> From a fluorescence scan it would appear a protein I am working on has
> zinc in it. The occupancy is likely to be very low however (a structural
> homologue has several zincs in the x-ray crystal data but at 0.5
> occupancy), as there isn't anything obvious in the electron density map
> (perhaps some of the waters are zinc) and an anomalous difference map
> wasn't possible to obtain on our last beamtime.
>
> Ideally I would want to re-express the protein with zinc added to the
> culture conditions, but I am time-restained, so I was wondering if it is
> possible to add zinc to purified protein instead? I have heard it can cause
> proteins to crash out. I have quite a lot of protein frozen so I can try a
> few things. I would appreciate any advice on how much to add from anyone
> who has had success with this before?
>
> Thanks in advance!
>
> 
>
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>



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Re: [ccp4bb] Adding Zinc to Protein

[Ivan Shabalin]

Hi Nicola,

I would just add zinc solution to the protein before the crystallization 
and/or to the drop with crystals.


An important thing to keep in mind is that Zn salt solutions will have 
low pH, unless a sufficient buffer is used.


I recommend having a look at the article

"Characterizing metal-binding sites in proteins with X-ray crystallography"

https://www.ncbi.nlm.nih.gov/pubmed/29674755

With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908
https://www.linkedin.com/in/shabalinig/
https://minorlab.org/person/ivan_s/

On 12/9/18 16:31, Nicola Evans wrote:

 From a fluorescence scan it would appear a protein I am working on has zinc in 
it. The occupancy is likely to be very low however (a structural homologue has 
several zincs in the x-ray crystal data but at 0.5 occupancy), as there isn't 
anything obvious in the electron density map (perhaps some of the waters are 
zinc) and an anomalous difference map wasn't possible to obtain on our last 
beamtime.

Ideally I would want to re-express the protein with zinc added to the culture 
conditions, but I am time-restained, so I was wondering if it is possible to 
add zinc to purified protein instead? I have heard it can cause proteins to 
crash out. I have quite a lot of protein frozen so I can try a few things. I 
would appreciate any advice on how much to add from anyone who has had success 
with this before?

Thanks in advance!



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Re: [ccp4bb] Adding Zinc to Protein

[Raghurama Hegde]

Hi Nicola, 

If all you are looking for is evidence that you have zinc in your structure 
based on the anomalous difference map, then with the data you already have you 
should be able to calculate the anomalous difference map! All you have to do is 
to reprocess the data in anomalous mode or whatever your favourite data 
processing software calls it. That will process the data with Friedel mates 
kept separate and you can get anomalous differences from them. If you have 
collected the data away from the absorption edge of zinc you should still be 
able to get anomalous differences though they might be small. 

What wavelength was used for the data collection? 

HTH 
Raghu

-Original Message-
From: CCP4 bulletin board  On Behalf Of Nicola Evans
Sent: Monday, December 10, 2018 03:02
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Adding Zinc to Protein

From a fluorescence scan it would appear a protein I am working on has zinc in 
it. The occupancy is likely to be very low however (a structural homologue has 
several zincs in the x-ray crystal data but at 0.5 occupancy), as there isn't 
anything obvious in the electron density map (perhaps some of the waters are 
zinc) and an anomalous difference map wasn't possible to obtain on our last 
beamtime. 

Ideally I would want to re-express the protein with zinc added to the culture 
conditions, but I am time-restained, so I was wondering if it is possible to 
add zinc to purified protein instead? I have heard it can cause proteins to 
crash out. I have quite a lot of protein frozen so I can try a few things. I 
would appreciate any advice on how much to add from anyone who has had success 
with this before? 

Thanks in advance!



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Re: [ccp4bb] Adding Zinc to Protein

[Olga Moroz]

Hi Nicola,

One way to do it is to dilute your protein, 10-100 times, and add zinc (also 
diluted), then concentrate.
Here is the procedure we used some time ago for a zinc-binding protein:

“S100A12 was diluted to 0.1 mg/ml-1 (approximately 10 mM) in a buffer 
containing 20 mM Tris-HCl pH 7.5, 200 mM NaCl and 10 mM zinc-acetate and 
concentrated to 10 mg/ml by ultrafiltration with 10 kDa cutoff membrane (Amicon 
Ultra 15, Millipore; Vivaspin 500ul, Sartorius Stedim Biotech). The procedure 
was repeated three times to achieve complete saturation with zinc while 
avoiding aggregation due to higher zinc concentrations”.

It worked, and we got a zinc complex :)

Good luck,

Olga


> On 9 Dec 2018, at 21:31, Nicola Evans 
> <251ca3b4615e-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> From a fluorescence scan it would appear a protein I am working on has zinc 
> in it. The occupancy is likely to be very low however (a structural homologue 
> has several zincs in the x-ray crystal data but at 0.5 occupancy), as there 
> isn't anything obvious in the electron density map (perhaps some of the 
> waters are zinc) and an anomalous difference map wasn't possible to obtain on 
> our last beamtime. 
> 
> Ideally I would want to re-express the protein with zinc added to the culture 
> conditions, but I am time-restained, so I was wondering if it is possible to 
> add zinc to purified protein instead? I have heard it can cause proteins to 
> crash out. I have quite a lot of protein frozen so I can try a few things. I 
> would appreciate any advice on how much to add from anyone who has had 
> success with this before? 
> 
> Thanks in advance!
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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Re: [ccp4bb] Adding Zinc to Protein

[Sheena McGowan]

Hi Nicola,

We have had success simply soaking zinc into the crystal prior to data 
collection. This has worked very well for a number of proteins. We simply add 
some zinc to the cryo-protectant and leave it to soak for various times.

Hope this helps.

Kind regards
Sheena







Sheena McGowan
Head, Structural Microbiology Laboratory
Monash Biomedicine Discovery Institute and Department of Microbiology
Adjunct Senior Research Fellow | Monash Institute of Pharmaceutical Sciences
Monash University 
Rm 137, Building 76, Wellington Rd, Clayton
Victoria, AUSTRALIA 3800
T + 61 3 9902 9309 | M +61419399454
E sheena.mcgo...@monash.edu  | Skype s.mcgowan

Dept webpage  | 
Bio/Publications/Projects 

 | Lab Facebook Page  | 
ORCID  | Google Scholar 
 |

Lorne Conference for Protein Structure and Function 
10th - 14th February, 2019

I acknowledge the Traditional Owners and Custodians of the lands on which I 
live and work and pay my respects to Elders past, present and future.

> On 10 Dec 2018, at 8:31 am, Nicola Evans 
> <251ca3b4615e-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> From a fluorescence scan it would appear a protein I am working on has zinc 
> in it. The occupancy is likely to be very low however (a structural homologue 
> has several zincs in the x-ray crystal data but at 0.5 occupancy), as there 
> isn't anything obvious in the electron density map (perhaps some of the 
> waters are zinc) and an anomalous difference map wasn't possible to obtain on 
> our last beamtime. 
> 
> Ideally I would want to re-express the protein with zinc added to the culture 
> conditions, but I am time-restained, so I was wondering if it is possible to 
> add zinc to purified protein instead? I have heard it can cause proteins to 
> crash out. I have quite a lot of protein frozen so I can try a few things. I 
> would appreciate any advice on how much to add from anyone who has had 
> success with this before? 
> 
> Thanks in advance!
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




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[ccp4bb] Adding Zinc to Protein

[Nicola Evans]

From a fluorescence scan it would appear a protein I am working on has zinc in 
it. The occupancy is likely to be very low however (a structural homologue has 
several zincs in the x-ray crystal data but at 0.5 occupancy), as there isn't 
anything obvious in the electron density map (perhaps some of the waters are 
zinc) and an anomalous difference map wasn't possible to obtain on our last 
beamtime. 

Ideally I would want to re-express the protein with zinc added to the culture 
conditions, but I am time-restained, so I was wondering if it is possible to 
add zinc to purified protein instead? I have heard it can cause proteins to 
crash out. I have quite a lot of protein frozen so I can try a few things. I 
would appreciate any advice on how much to add from anyone who has had success 
with this before? 

Thanks in advance!



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https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1